Download Ribonuclease P（Human）Real Time RT-PCR Kit User

Revision No.: ZJ0004
Issue Date: Jul 1st, 2012
Ribonuclease P（Human）Real Time RT-PCR Kit
User Manual
For In Vitro Diagnostic Use Only
QR-0142-02
For use with ABI Prism®7000/7300/7500/7900/Step One Plus; iCycler iQ™4/iQ™5;
Smart Cycler II;Bio-Rad CFX 96;Rotor Gene™6000; Mx3000P/3005P;MJ-Option2/Chromo4;
LightCycler®480 Instrument
Obelis S.A.
Boulevard Général Wahis 53
1030 Brussels, BELGIUM
Tel: +(32) 2.732.59.54
Fax: +(32) 2.732.60.03
E-Mail : [email protected]
Shanghai ZJ Bio-Tech Co., Ltd.
www.liferiver.com.cn
Tel: +86-21-34680596
[email protected]
Fax: +86-21-34680595
2nd floor,No.15 Building,No.188 Xinjunhuan Road,
PuJiang Hi-tech Park, Shanghai, China
1. Intended Use
Ribonuclease P real time RT-PCR Kit is used for the detection of Ribonuclease P in human samples
(blood, tissue, and etc.) by using real time PCR systems.
2. Principle of Real-Time PCR
The principle of the real-time detection is based on the fluorogenic 5’nuclease assay. During the PCR
reaction, the DNA polymerase cleaves the probe at the 5’ end and separates the reporter dye from the
quencher dye only when the probe hybridizes to the target DNA. This cleavage results in the
fluorescent signal generated by the cleaved reporter dye, which is monitored real-time by the PCR
detection system. The PCR cycle at which an increase in the fluorescence signal is detected initially is
proportional to the amount of the specific PCR product. Monitoring the fluorescence intensities in
real-time allows the detection of the accumulating product without having to re-open the reaction tube
after the amplification.
3. Product Description
All living things synthesize an enzyme — called Ribonuclease P (Rnase P) — that cleaves the head (5')
end of the precursors of transfer RNA (tRNA) molecules.
Ribonuclease P real time RT-PCR kit contains a specific ready-to-use system for the detection of the
RnaseP in human specimens using RT-PCR (Reverse Transcription Polymerase Chain Reaction) in the
real-time PCR system. The master contains Super Mix for the specific amplification of RnaseP. The
reaction is done in one step real time RT-PCR. The first step is a reverse transcription (RT), during
which the RnaseP is transcribed into cDNA. Afterwards, a thermostable DNA polymerase is used to
amplify the specific gene fragments by means of PCR (polymerase chain reaction). Fluorescence is
emitted and measured by the real time systems optical unit during the PCR. The detection of amplified
RnaseP fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQ1. In
addition, the kit contains a system to identify possible PCR inhibition by measuring the HEX/VIC/JOE
fluorescence of the internal control (IC). An external positive control defined as 1×107copies/ml is
supplied which allow the determination of the gene load. For further information, please refer to
section 9.2 Quantitation.
4. Kit Contents
Ref.
Type of reagent
Presentation 25rxns
1
RnaseP Super Mix
1 vial, 480ml
2
RT-PCR Enzyme Mix
1 vial, 28ml
3
Molecular Grade Water
1 vial, 400µl
7
4
RnaseP Positive Control(1×10 copies/ml)
1 vial, 30µl
3
3
Analysis sensitivity: 1×10 copies/ml
LOQ: 2×10 ～1×108copies/ml
Note: Analysis sensitivity depends on the sample volume, elution volume, nucleic acid extraction
methods and other factors .If you use the RNA extraction kits recommended, the analysis sensitivity is
the same as it declares. However, when the sample volume is dozens or even hundreds of times greater
than elution volume by some concentrating method, it can be much higher.
5. Storage
• All reagents should be stored at -20°C. Storage at +4°C is not recommended.
• All reagents can be used until the expiration date indicated on the kit label.
• Repeated thawing and freezing (> 3x) should be avoided.
• Cool all reagents during the working steps.
• Super Mix and Reaction Mix should be stored in the dark.
6. Additionally Required Materials and Devices
• Biological cabinet
• Real time PCR system
• Vortex mixer
• Real time PCR reaction tubes/plates
• Cryo-container
• Pipets (0.5µl – 1000µl)
• Sterile filter tips for micro pipets
• Sterile microtubes
• Disposable gloves, powderless
• Biohazard waste container
• Refrigerator and Freezer
• Tube racks
• Desktop microcentrifuge for “eppendorf” type tubes (RCF max. 16,000 x g)
7.
Warnings and Precaution
• Carefully read this instruction before starting the procedure.
• For in vitro diagnostic use only.
• This assay needs to be carried out by skilled personnel.
• Clinical samples should be regarded as potentially infectious materials and should be prepared in
a laminar flow hood.
• This assay needs to be run according to Good Laboratory Practice.
• Do not use the kit after its expiration date.
• Avoid repeated thawing and freezing of the reagents, this may reduce the sensitivity of the test.
• Once the reagents have been thawed, vortex and centrifuge briefly the tubes before use.
• Prepare quickly the Reaction mix on ice or in the cooling block.
• Set up two separate working areas: 1) Isolation of the RNA/ DNA and 2) Amplification/
detection of amplification products.
• Pipets, vials and other working materials should not circulate among working units.
• Use always sterile pipette tips with filters.
• Wear separate coats and gloves in each area.
• Do not pipette by mouth. Do not eat, drink, smoke in laboratory.
• Avoid aerosols.
8. Sample Collection, Storage and transport
• Collected samples in sterile tubes.
• Specimens can be extracted immediately or frozen at -20°C to -80°C.
9. Procedure
9.1 RNA-Extraction
RNA extraction kits are available from various manufacturers. You may use your own extraction
systems or the commercial kit based on the yield. For the RNA extraction, please comply with the
manufacturer’s instructions. The recommended extraction kit is as follows:
Nucleic Acid Isolation Kit
Cat. Number
Manufacturer
RNA Isolation Kit
ME-0010/ME-0012
ZJ Biotech
QIAamp Viral RNA Mini extraction Kit (50)
52904
QIAGEN
9.2 Quantitation
The kit can be used for quantitative or qualitative real-time RT-PCR.
For performance of quantitative real-time PCR, standard dilution must be prepared first as
follows. Molecular Grade Water is used for dilution.
Dilution is not needed for performance of qualitative real-time PCR.
Take positive control (1×107copies/ml) as the starting high standard in the first tube. Respectively
pipette 36ul of Molecular Grade Water into next three tubes. Do three dilutions as the following
figures:
To generate a standard curve on the real-time system, all four dilution standards should be used and
defined as standards with specification of the corresponding concentrations.
Attention:
A. Mix thoroughly before next transfer.
B. The positive control (1×107copies/ml) contains high concentration of the target DNA. Therefore, be
careful during the dilution in order to avoid contamination.
9.3 RT-PCR Protocol
The Master Mix volume for each reaction should be pipetted as follows:
1)
2)
3)
4)
5)
The volumes of Super Mix and Enzyme Mix per reaction multiply with the number of samples,
which includes the number of controls, standards, and sample prepared. Molecular Grade Water
is used as the negative control. For reasons of unprecise pipetting, always add an extra virtual
sample. Mix completely then spin down briefly in a centrifuge.
Pipet 20µl Master Mix with micropipets of sterile filter tips to each of the real time PCR
reaction plate/tubes. Separately add 5µl RNA sample template, positive and negative controls to
different plate/tubes. Immediately close the plate/tubes to avoid contamination.
Spin down briefly in order to collect the Master Mix in the bottom of the reaction tubes.
Perform the following protocol in the instrument:
45°C for 10min
1cycle
Selection of fluorescence channels
95°C for 15min
1cycle
FAM
Target Nucleic Acid
95°C for 15sec, 60°C for 1min
40cycles
( Fluorescence measured at 60°C)
If you use ABI Prism® system, please choose “none” as passive reference and quencher.
10. Threshold setting: just above the maximum level of molecular grade water.
11. Calibration for quantitative detection: Input each concentration of standard controls at the end
of run, and a standard curve will be automatically formed.
12. Quality control:
Negative control, positive control, internal control and QS curve must be performed correctly,
otherwise the sample results is invalid.
Channel
Ct value
Control
FAM
Molecular Grade Water
UNDET
Positive Control(qualitative assay)
≤35
QS（quantitative detection）
Correlation coefficient of QS curve≤－0.98
13. Data Analysis and Interpretation
The following sample results are possible:
Ct value
Result Analysis
1#
UNDET
It can be considered a failure of RNA extraction from the sample
2#
≤38
it can be considered a success of RNA extraction from the sample
3#
Re-test; if it is still 38~40, report as 1#
38～40
For further questions or problems, please contact our technical support at [email protected]